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Application of FISH in hematologic malignancies

Application of FISH in hematologic malignancies. Dr Edmond S K Ma Department of Pathology Hong Kong Sanatorium & Hospital. Molecular Cytogenetics.

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Application of FISH in hematologic malignancies

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  1. Application of FISH in hematologic malignancies Dr Edmond S K Ma Department of Pathology Hong Kong Sanatorium & Hospital BTG 2013

  2. Molecular Cytogenetics • The utilization of techniques based on fluorescence in-situ hybridization in which DNA probes are labelled with different fluorochromes to map one or more specific regions of the genome • Bridges cytogenetics and molecular genetics • Techniques: • FISH • CGH • 24-colour karyotyping (M-FISH / SKY) • Array CGH BTG 2013

  3. Any role for FISH in the post-genomic era? • Manageable by routine diagnostic laboratories • Answer to specific clinical questions • Practical advantages • Numerical abnormality • Multiple fusion partners • Breakpoint heterogeneity • Applicable to many specimen types BTG 2013

  4. Probes Chromosome enumeration Locus specific Orange signal: chr 1; Green signal: chr 7 BCR-ABL dual colour dual fusion Multicolour FISH Chromosome painting der(9) dic(14;22)der(22)

  5. FISH as an investigative tool in haematological malignancies • Detection of numerical and structural abnormalities in interphase and metaphase cells • Characterization of marker chromosomes • Detection of cryptic translocation • Usually detected by CG • Not usually detected by CG • Lineage involvement by the neoplastic clone • Disease monitoring after treatment • Chimerism study post-sex-mismatched BMT BTG 2013

  6. From Ma, Wan & Chan. Cancer Reviews Asia-Pacific 2: 131 – 141, 2004

  7. Acute promyelocytic leukaemia (APL) with unusual CG Wan TS et al, Cancer Genet Cytogenet 121: 90 – 3, 2000 BTG 2013

  8. Wan TS et al, Cancer Genet Cytogenet 121: 90 – 3, 2000

  9. Cryptic insertion of BCR at 9q34 in CML D-FISH: 1R2G1F pattern D-FISH S-FISH ES-FISH Wan TS et al, Leukemia 18: 161 – 2, 2004

  10. Chimerism status by XY-FISH BTG 2013

  11. Chronic myeloid leukaemia post-BMT donor relapse BTG 2013

  12. FISH: some advantages • Genetic abnormality measurable in dividing and non-dividing cells • Covers CG failure • Covers mature B-cell disorders • Applicable to many specimen types • Applicable to heterogeneous breakpoints or multiple translocation partners • Quantitative • Standardization • Nomenclature (ISCN), criteria for interpretation and proficiency testing BTG 2013

  13. MLL probe for rearrangement BTG 2013

  14. Characterization of chromosome 11q deletion Ma SK et al, Leukemia 16: 953 – 955, 2002 BTG 2013

  15. Southern Blot hybridization for MLL rearrangement Ma SK et al, Leukemia 16: 953 – 955, 2002 BTG 2013

  16. Caveats of FISH analysis • No global view of chromosomal complement • Requires clinicopathological or prior cytogenetics information • Issues related to analytical sensitivity and probe specificity • Susceptibility to artifacts • Cannot detect minute aberrations (< 20 kb) • Aneuploidy versus amplification BTG 2013

  17. Ph chromosome Chronic myeloid leukaemia BTG 2013

  18. From Ma, Wan & Chan. Cancer Reviews Asia-Pacific 2: 131 – 141, 2004

  19. BCR-ABL dual colour single fusion translocation probe

  20. Detection of fusion genes by S-FISH BTG 2013

  21. Detection of BCR-ABL gene fusion by S-FISH • Accurate for metaphase FISH • Problem of false positive (~ 4%) • Normal cutoff range • 10% (Dewald et al, Cancer Genet Cytogenet 71: 7; 1993) • 7% (Cox Froncillo et al, Ann Hematol 73: 113; 1996) BTG 2013

  22. Detection of fusion genes by ES-FISH

  23. Detection of fusion genes byES-FISH BTG 2013

  24. BCR-ABL dual colour dual fusion translocation probe

  25. BCR-ABL dual fusion translocation probe BTG 2013

  26. Detection of BCR-ABL gene fusion by D-FISH • Normal range for 500 interphase nuclei •  4 nuclei ( 0.8%) • Buño et al, Blood 92: 2315; 1998 • Monitor response to therapy • Normal cutoff for 6,000 nuclei = 0.079% • Residual disease level 7 - 53 nuclei (0.117 - 0.883 %) • Dewald et al, Blood 91: 3357; 1998 BTG 2013

  27. Three-way Ph translocation *Courtesy of Dr. K. F. Wong, QEH BTG 2013

  28. Variant D-FISH pattern BTG 2013

  29. Derivative chromosome 9 (9q+) deletion in CML • Occurs in ~ 15% of cases • Deletion of reciprocal ABL-BCR fusion gene • At the time of Ph translocation • Correlates with a poor prognosis • Sinclair et al. Blood 95: 738 - 743, 2000 • Huntly et al. Blood 98: 1732 - 1738, 2001 • Partly overcome by imatinib • Huntly et al. Blood 102: 2205 – 2212, 2003 BTG 2013

  30. 9 22 der(9) der(22) Derivative chromosome 9 deletion in CML Confirmation: >10% of cells S-FISH Metaphase FISH RT-PCR Wan TS et al, J Clin Pathol 56: 471 – 474, 2003

  31. Primo et al, 2003 83% typical 17% atypical Wan et al, 2003 Among 46 CML Typical = 44 (95%) Atypical = 2 Lisa Siu (QEH, 2008) Among 22 CML Typical = 17 (77%) ABL-BCR deletion = 2 ABL deletion = 2 BCR deletion = 1 Atypical BCR-ABL interphase D-FISH patterns BTG 2013

  32. BCR-ABL + 9q34 tricolour dual fusion translocation probe Normal cell: 2 G + 2 O/aqua Ph+ cell: 1 G + 1 O/aqua + 1 G/O fusion + 1 G/O/aqua fusion der(9) deletion cell: 1 G + 1 O/aqua + 1 G/O fusion False+ cell: 1 G + 1 O/aqua + 1 G/O/aqua fusion BTG 2013

  33. BCR-ABL + 9q34 tricolour dual fusion translocation probe Normal cell: 2 G + 2 O/aqua Ph+ cell: 1 G + 1 O/aqua + 1 G/O fusion + 1 G/O/aqua fusion der(9) deletion cell: 1 G + 1 O/aqua + 1 G/O fusion False+ cell: 1 G + 1 O/aqua + 1 G/O/aqua fusion BTG 2013

  34. der(9) deletion BCR-ABL D-FISH BCR-ABL + 9q34 tricolour dual fusion translocation probe fusion fusion BTG 2013

  35. Clinical use of interphase FISH in risk stratification • CLL • 13q-, 11q-, 17p-, +12 • Myeloma • High-risk cytogenetic markers • t(4;14) • t(14;16) • del(17)p/p53 • chromosome 1q gain • Coupled with cell sorting or immunofluorescence BTG 2013

  36. FISH and personalized medicine • Myeloma • CLL • Imatinib targets • BCR-ABL • FIP1L1-PDGFRa fusion • PDGFRb rearrangements • MDS • 5q- BTG 2013

  37. BTG 2013

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